Cells (1×105/ml) were fixed in 4% paraformaldehyde for 20 min at room temperature and permeabilized using 0.1% Triton X-100 for 20 min. Samples were blocked in goat serum (Wuhan Boster Biological Technology, Ltd.; cat. no. AR009) for 30 min and subsequently incubated with the following primary antibodies: Anti-β-catenin (Wanleibio Co., Ltd.; cat. no. WL0962a; 1:200) and anti-mono-ADP-ribose binding reagent (EMD Millipore; cat. no. MABE1076; 1:200) at 4°C overnight. The following day, cells were washed in PBS and incubated with Cy3-conjugated secondary antibody (ProteinTech Group, Inc.; cat. no. SA00009-2; 1:200) in the dark for 1 h at room temperature. DAPI (Wuhan Boster Biological Technology, Ltd.) was used for nuclear staining (5 min at room temperature). The images were captured using ZOETM Fluorescent Cell Imager (Bio-Rad Laboratories, Inc.) and analyzed using ImageJ software (version 1.48; National Institutes of Health) The experiment was repeated three times.
Anti mono adp ribose binding reagent
Manufactured by Merck Group
Sourced in Germany
The Anti-mono-ADP-ribose binding reagent is a laboratory tool designed to detect and study the presence of mono-ADP-ribosylation, a post-translational modification of proteins. This reagent specifically binds to and recognizes mono-ADP-ribosylated proteins, enabling their identification and analysis.
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Lab products found in correlation
Anti rabbit hrp antibody, by Bio-Rad (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Goat serum, by Wuhan Boster Biological Technology (1 mentions)
Cy3 conjugated secondary antibody, by Proteintech (1 mentions)
Zoetm fluorescent cell imager, by Bio-Rad (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Boster Biological Technology (1 mentions)
Rabbit polyclonal anti actin antibody, by Merck Group (1 mentions)
Rabbit polyclonal anti histone h3, by Abcam (1 mentions)
Anti α tubulin, by Thermo Fisher Scientific (1 mentions)
Protein g dynal beads, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
6 protocols using anti mono adp ribose binding reagent
1
Immunofluorescent Analysis of β-Catenin and ADP-Ribose
2
Actin-dependent ADP-ribosylation Assay
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1 μg of actin (in G-, or F- state, with latrunculin or phalloidin, if indicated) in the assay buffer (1 mM NAD+, 20 mM Tris pH 8, 150 mM NaCl, and 1 mM MgCl2) was added to the indicated amount of TcHVR or Iota-A in the total volume of 10 μl. After 10 minutes of incubation at 37 °C, the reaction was stopped by adding Laemmli sample buffer (62.5 mM Tris-HCl pH 8.0, 25 mM DTT, 1.5% SDS, 10% glycerol, and 0.1 mg/ml bromphenol blue) and heating the sample at 95 °C for 5 minutes. Components of the mixture were separated by SDS-PAGE, blotted onto a polyvinylidene difluoride membrane using a Trans-Blot Turbo Transfer System, and visualized using a combination of anti-mono-ADP-ribose binding reagent (dilution 1 to 5000, MABE1016 Merck) and anti-rabbit-HRP antibody (dilution 1 to 5000, #1706515, Bio-Rad).
3
Actin ADP-Ribosylation Assay Protocol
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A total of 8-μl mixtures of 1 μg (2.4 μM) actin and Lifeact peptide at specified concentrations were preincubated for 5 minutes at room temperature in TccC3 buffer (1 mM NAD, 20 mM Tris pH 8, 150 mM NaCl, and 1 mM MgCl2). The ADP-ribosylation reaction was initiated by addition of 0.02 μg (61 pM) of TccC3HVR into the mixture. After 10 minutes of incubation at 37°C, the reaction was stopped by adding Laemmli sample buffer (62.5 mM Tris-HCl pH 8.0, 25 mM DTT, 1.5% SDS, 10% glycerol, and 0.1 mg/ml bromphenol blue) and heating the sample at 95°C for 5 minutes. Components of the mixture were separated by SDS-PAGE, blotted onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blot Turbo Transfer System (Bio-Rad), and visualized using a combination of anti-mono-ADP-ribose binding reagent (Merck, Germany) and anti-rabbit-HRP antibody (Bio-Rad). The level of actin ADP-ribosylation was quantified by densitometry using Image Lab software (Bio-Rad).
4
Actin ADP-Ribosylation Assay
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8 µl mixtures of 1 µg (2.4 µM) actin and Lifeact peptide at specified concentrations were preincubated for 5 minutes at room temperature in TccC3 buffer (1 mM NAD, 20 mM Tris pH 8, 150 mM NaCl and 1 mM MgCl2). The ADP-ribosylation reaction was initiated by addition of 0.02 µg (61 pM) of TccC3HVR into the mixture. After 10 minutes of incubation at 37 °C, the reaction was stopped by adding Laemmli sample buffer and heating the sample at 95 °C for 5 minutes. Components of the mixture were separated by SDS-PAGE, blotted onto a PVDF membrane using a Trans-Blot Turbo transfer system (Bio-Rad) and visualized using a combination of anti-mono-ADP-ribose binding reagent (Merck) and anti-rabbit-HRP antibody (Bio-Rad). The level of actin ADP-ribosylation was quantified by densitometry using Image Lab software (Bio-Rad).
5
Plasmid Constructs and Antibody Details
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The plasmid encoding His-fused full length human PARP1 was a gift from John Pascal and described elsewhere [52 (link), 53 (link)]. The plasmid encoding the HA-fused full length human EZH2 plasmid (pCMVHA hEZH2) was a gift from Kristian Helin (Addgene plasmid # 24230) [54 (link)]. The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55 (link)]. All plasmids were verified by sequencing. Rabbit polyclonal anti-His (WB 1/200, Ip: 2.5 μgr/sample), rabbit anti-Mouse IgG HRP conjugated (WB: 1/5000; IP 2.5 μgr/sample), and mouse anti-Rabbit IgG HRP conjugated (WB: 1/5000, IP: 2.5 μgr/sample) were from Santa Cruz Biotechnology. Monoclonal mouse anti-HA (WB 1/1000) was from Origene. Monoclonal mouse anti-HA (IP 2.5 μgr/sample) and rabbit polyclonal anti-Histone H3 (WB 1/2000) were from Abcam. Rabbit polyclonal anti-PARP1 (WB 1/10000) and rabbit polyclonal anti-H3K27me3 (WB 1:2000) were from Active Motif. Rabbit polyclonal anti-PAR antibody (WB 1/1000) was from Trevigen. Mouse monoclonal anti-EZH2 antibody (WB 1/2000; IP 2.5 μgr/sample) was from BD Bioscience. Rabbit polyclonal anti-Actin antibody (WB 1/100) was from Sigma Aldrich. Anti-mono-ADP-ribose binding reagent and Anti-mono- and poly-ADP-ribose binding reagent (IP 5 μgr/sample) were from Millipore.
6
Verification of α-Tubulin Mono-ADP-Ribosylation
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Mono-ADP-ribosylation (MAR) of α-tubulin was verified by immunoprecipitation as previously described (Hutin et al., 2018a (link)). In brief, mNSCs were harvested and the cell pellet was lysed in cell lysis buffer mNSCs (200 mM NaCl, 1% NP40, and 20 mM HEPES at pH 7.4, supplemented with protease inhibitor cocktail; Sigma). Lysates were clarified by centrifugation and the supernatant was incubated for 2 h with anti- α-tubulin (Sigma) antibody and protein G Dynal beads (Thermo Fisher). The bead-antibody-protein complex was then washed three times for 5 min in wash buffer (200 mM NaCl, 0.1% NP40, and 20 mM HEPES at pH 7.4, supplemented with protease inhibitor cocktail; Sigma). The proteins were then eluted from the beads with 2× Laemmli buffer and separated by SDS–PAGE and transferred to PVDF membranes. Membranes were probed with anti-mono-ADP-ribose-binding reagent (Millipore) followed by incubation with the appropriate secondary antibody. A list of all antibodies is provided in Table 2 .
COS-1 cells were transfected with 2 μg of EGFP, GFP-Tiparp, or GFP-TiparpH532A plasmids (MacPherson et al., 2013 (link)) per well of a six-well plate using Lipofectamine 2000 reagent according to the manufacturer’s instructions. The next morning, cells were harvested, protein extracts were made and the immunoprecipitation was conducted as described above.
COS-1 cells were transfected with 2 μg of EGFP, GFP-Tiparp, or GFP-TiparpH532A plasmids (MacPherson et al., 2013 (link)) per well of a six-well plate using Lipofectamine 2000 reagent according to the manufacturer’s instructions. The next morning, cells were harvested, protein extracts were made and the immunoprecipitation was conducted as described above.
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